For applications in biodefense, it is desirable for small molecule inhibitors to target multiple category A-C agents, because it is difficult and expensive to develop even one small molecule inhibitor. Relatively few clinically useful drugs are active against multiple different pathogens. Of the few that are, a substantial fraction target DMA modifying enzymes. Examples include topoisomerase inhibitors used as antimicrobials against many bacteria, and nucleotide reverse transcriptase inhibitors used against HIV and HBV. In our previous MARCE-funded projects, we have initiated development of therapeutics for two DMA modifying enzymes that are found in multiple category A-C agents. One is a type 1B topoisomerase, the second a Holliday junction resolvase. Initial studies focused on examples of these enzymes found in poxviruses. We have established assays in vitro and used them to screen -225,000 small molecules for inhibitory activity at the Northeast RCE Screening Facility and at Merck Research Laboratories. We have also carried out background mechanistic studies to inform our efforts at inhibitor design. Results include 1) collaborating to solve the structure of the variola topoisomerase enzyme bound to DMA and 2) revising our understanding of favored substrates for the vaccinia resolvase. We have identified molecules with activity against each purified protein in vitro and against poxvirus replication in cell culture assays. We propose to use these as starting points to find inhibitors active against both poxviruses and Coccidioides, the fugus responsible for Valley Fever, which was recently added to the select agent list and encodes a resolvase resembling the poxvirus enzyme. Going forward, we will develop our lead inhibitors and carry out mechanistic studies to aid inhibitor development. This project is intended for Research Program I, since it concerns the use of small molecule inhibitors to modulate interactions of Emerging Viruses with Host Cell Pathways, but we also expect to have extensive interactions with programs IV and VI.